Recently an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was isolated from rabbit brain which catalyzes fusion of membrane of membranes containing plasmenylethanolamine lipids, which readily adopt an inverted hexagonal phase. Here, we demonstrate that this GAPDH isoform can catalyze fusion between naturally occurring subcellular membranes involved in insulin exocytosis. With purified pancreatic islet b-cell plasma membranes and secretory granules, the GAPDH isoform catalyzes fusion of the two membrane populations in a manner that is inhibited by monoclonal antibodies directed against GAPDH. Both membrane compartments were required, and the GAPDH isoform did not catalyze fusion of one secretory granule population with another. HIT insulinoma cells were demonstrated to contain a similar fusion-catalyzing activity in cytosol. The fusion factors failed to catalyze fusion of membranes which had been depleted of plasmenylethanolamine species by exposure to acid. Such treatment has been demonstrated by electrospray ionization tandem mass spectrometry to destroy the plasmenylethanolamine species in these membranes.